Microbial digestion of tobacco materials

ABSTRACT

Microbial digestion of tobacco is disclosed wherein a slurry of the pectin bound tobacco plant matter is treated with an inoculum containing pectolytic-enzyme producing organisms selected from the class of Erwinia carotovora, Erwinia atroseptica, Erwinia aroideae, Bacillus polymyxa and Streptomyces cellulosae. The slurry is maintained at a pH of 5.2 to 8.5 under proper environmental conditions to organism growth and resultant fibrillation. Termination of the organism activity is effected by heating and the final step involves sheeting of thickened slurry.

United States Patent [1 1 Gravely et al.

[ July 24, 1973 MICROBIAL DIGESTION OF TOBACCO MATERIALS [73] Assignee:Brown & WilliamsonTobneco Corporation, Louisville, Ky.

[22] Filed: June 18, 1971 [21] Appl. No.: 154,624

[52] US. Cl. .1 131/141 [51] Int. Cl. A24b 03/14, A24b 15/00 [58] Fieldof Search ..131/140,141,17;

[56] References Cited UNITED STATES PATENTS 3,240,214 3/1966 Bavley eta1. 131/141 1,331,331 2/1920 Erslev 131/141 FOREIGN PATENTS ORAPPLICATIONS 1,153,120 5/1969 Great Britain 131/141 OTHER PUBLICATIONSKertesz, Z. 1., The Pectic Substances(Text) Published by IntersciencePublishers Inc. N. Y. (I951) Pages 587, 588 and 589 cited.

Bergeys Manual of Determinative Bacteriology (text) by Breed, Murray andSmith -Seventh Edition (1957) Pub. by The Williams and Wilkins Co.Baltimore, Md. pages 355-358 inc. Cited.

Primary Examiner-Melvin D. Rein Attorney-Kane, Dalsimer, Kane, Sullivan& Kurucz [5 7] ABSTRACT Microbial digestion of tobacco is disclosedwherein a slurry of the pectin bound tobacco plant matter is treatedwith an inoculum containing pectolytic-enzyme producing organismsselected from the class of Erwinia carotovora, Erwinia atroseptica,Erwinia aroideae, Bacillus polymyxa and Streptomyces cellulosae. Theslurryis maintained at a pH of 5 .2 to 8.5 under proper environmentalconditions to organism growth and resultant fibrillation. Termination ofthe organism activity is effected by heating and the final step involvessheeting of thickened slurry.

3 Claims, No Drawings MICROBIAL DIGESTION OF TOBACCO MATERIALSBACKGROUND OF THE INVENTION In the utilization of plant material,particularly where thin sheets are formed therefrom, such asreconstituted tobacco, particles of the plant material are fibrillatedby some mechanical work input means such as beaters, homogenizersor thelike. Installations which include work input means to fibrillateparticles of plant material are expensive to install, operate andmaintain. Where the plant material used is tobacco, generally only themanufacturing waste is employed. Tobacco manufacturing waste consists ofvarious portions of tobacco including particles of lamina, veins, ribs,stems or tobacco dust. All of this material, although useful, is toosmall or too coarse to be incorporated into a tobacco product. Thestems, veins and ribs because of their cellular structure require aconsiderable amount of mechanical work input to break their cellularstructure so that small bundles of fibrils are released therefrom. Inthe fibrillous form, the stems, veins and ribs may be readilyincorporated into the reconstituted tobacco process with little or nofurther mechanical work. i

It is an object of the invention herein to provide a process in whichpectin-bound plant material is fibrillated'without the substantial useof mechanical work input means.

lt is also an object of the inventionto provide microbial means forfibrillating pectin-bound plant material such as tobacco whilemaintaining the natural flavor and aroma thereof.

THE INVENTION The invention generally contemplates the microbialdigestion or disintegration of pectin-bound plant materials, forexample, tobacco or other fibrous plant material wherein pectin, theintercellular cement that binds cells to cells and fibers to fibers isbroken down so as to separate and form individual plant fibrils forsubsequent processing. Plant material, for example tobacco, is subjectedto the action of a pectolytic enzyme producing microorganism undercontrolled conditions of thermal environment, pH, time and moisture. Themicrobial digestion or disintegration of the pectin-bound plant materialis completed after the plant parts exhibit extensive and substantiallycomplete fiber separation. The microbial action is ceased and the plantparts in fibrous form are ready for further processing and treatment.

It has been found that various species of pectolytic enzyme producingmicroorganisms may be employed for the microbial digestion ordisintegration of pectinbound plant material. Examples of the selectedgenera are Erwinia, Bacillus and Streptomyces. The following are speciesthat have been found to act on plant material to fibrillate thepectin-bound plant parts and obviate the necessity of employingmechanical work input means to accomplish substantial fibrillationthereof.

Genera Species ATCC No. Erwinia caromvora 495 Erwinia caralovora I38Erwim'a carotovora l 7799 Erwinia caromvara 8061 Erwinia carotovara15713 Erwinia atmseptica 4446 Erwinia alroseptica 4446 Erwinia aroideael2286 Erwinia araideae 123 12 Bacillus polymyxa 842 S treptom ycescellulosae 3 3 l 3 ATCC American Type Culture Collection AccessionNumber.

When employing one or more of the microorganisms from the species listedabove, it has been found convenient to employ standard media for growingthe microorganisms, then when maximum growth of the microorganisms isachieved, the medium containing the microorganisms, i.e., inoculum, isadded to the pectin-bound plant material in the presence of sufficientmoisture for maintaining optimum growth under controlled conditions ofpH, temperature and time.

In the preferred embodiments of the invention herein the pectin-boundplant material employed is tobacco. Various forms of the tobacco invarying degrees and stages of curing may be employed, for example,unredried flue-cured or burley strips, :redried flue-cured or burleystrips, burley stems, flue-cured stems, manufacturing fines, stalks,shredded tobacco and mixtures thereof.

When practicing the invention herein, tobacco plant parts of varyingsizes particularly the stems, veins or ribs are employed. Since theaction of the pectolytic enzyme producing microorganisms will cause thefibrillation of these parts at a substantially uniform rate, particlesof lamina and tobacco dust which require little, if any, mechanical workinput means would be fibrillated in a shorter period of time. Toaccomplish a microbial digestion or disintegration of tobacco plantparts an inoculum is prepared from a suitable substrate using one of thespecies listed above. After the microorganism has grown under optimumconditions in the substrate, the inoculum containing viablemicroorganism is added to the tobacco plant parts such as un cookedstems, veins or ribs and either flue-cured or burley or mixtures thereofunder controlled conditions of pH and temperature. The inoculated plantparts which induce pectolytic enzyme formation and activity aresubjected to the action of the microorganisms for a sufficient period oftime until the disintegration of the parts is substantially completed.This is determined by the consistency and viscosity of the formedslurry. Thereafter, the fibrillated plant parts are mixed with otherfractions of tobacco such as particles of lamina and tobacco dust; Themixture is then sized by employing a screening procedure in which theparticles are forced through screens having accurate openings. The sizedmixture is then made into reconstituted tobacco sheet material.

It has been found that the conditions for effecting substantialfibrillation'of plant parts may vary greatly, i.e., depending upontemperature, pH, agitation and aeration. The digestion may be completedafter 24 hours or may be completed in from 4 to 6 hours under optimumconditions. The pH of the tobacco slurry is maintained between 5.2 and8.5, preferably from about 6.5 to 7. The temperature may vary from about24 to 40 C., preferably from about 28 to 32 C. The tobacco slurry formedmay vary greatly but is preferably maintained between 2 and 14' percentsolids content on a weight basis, but optimally is maintained at fromabout 6 to 10 percent solids on a weight basis. It has also been foundthat disintegration of the plant parts can be accelerated if theinoculate-d plant parts are aerated and agitated to maintain uniformsuspension of tobacco parts with constant mixing and contact with airfor optimizing growth conditions of the microorganisms.

To have a better understanding of the invention herein the followingexamples are provided as specific embodiments thereof. It should beunderstood that proportions of the various component materials andvariation of process steps and conditions may be made without departingfrom the teaching herein.

EXAMPLE 1 A. Preparation of Inoculum An extract from burley stems isprepared as follows:

25 g of burley stem is mixed in 250 ml water and is cooked in anautoclave for 25 minutes at 15 psig and 121 C. The resultant liquor isremoved and the volume is adjusted to original amount. 1.5 percent agaris added to the liquor and is meltedand dispensed into clean containers.The solidified extract of burley stem is sterilized for future use.

A broth of the above extract may be prepared by omitting the addition ofagar as listed above. A nutrient dextrose broth (NDB) may also beprepared as follows:

Nutrient broth 8 gm Dextrose 10 gm Distilled water 1000 ml Obviously themedia or substrate for growing the particular microorganisms may varygreatly and, therefore, many changes in substrate or combinationsthereof are applicable. Maintenance of the microorganisms, particularlythe Erwinia carotovora species, has been successfully conducted ontryptone-glucose extract agar slants. These cultures were incubated for30 to 72 hours at 30 C. prior to use. Liquid media, for example NDBmedia, was inoculated with physiological salt (0.85 percent) washingsfrom slants, diluted to an optical density of 0.4 as read at 650 my. ona spectrophotometer. 2.5 ml of the standardized suspension was added to250 ml of the liquid broth media (NDB) for culture propagation. Bestgrowth was achieved by employing rotary agitation for 24 hours at 30 C.

B. Microbial Digestion of Tobacco A water tobacco mixture consisting of10 percent solids by weight was inoculated with the 10 percent by volumeof NDB cultures noted above. The tobaccomicroorganism mixture wasmaintained in a thermal environment of 30 C. for 16 hours and wascontinuously agitated. The pH of the mixture was maintained between 5.0and 7.0. After 16 hours the tobacco suspension was substantiallyfibrillated and was added into the reconstituted tobacco process. Thetreated tobacco mixture, including the liquor, was then heated to about190 F. to cease all bacterial enzymatic action. The slurry was thensized by passing through screens having an opening of from 0.016 to0.012 inch. The sized slurry was then deaerated and cast into areconstituted tobacco sheet.

EXAMPLE 2 50 grams of burley stem (1/2 to 1-1/2 inches long) was dividedinto two equal parts. Each part, consisting of 25 grams each, was mixedwith 250 ml of tap water in a 500 ml Erlenmeyer flask. into one flaskwas placed 25 ml of a 24 hour culture of Erwinia carotovora, ATCC495 (EC495) grown in Nutrient Dextrose Broth (NDB). Both flasks with andwithout EC 495 added were incubated under rotary agitation for 24 hoursat ambient temperature. The starting pH of both mixtures beforetreatment was 5.4. After 24 hours the mixture containing the EC 495,stem and water was observed to be very viscous in appearance with thestem fiber bundles being well separated. The flask which did not containEC 495 inoculum was observed to contain a marked predominance of intactwhole stem. After 24 hours the pH of the uninoculated mixture was 5.8while the pH of the inoculated mixture was 6.8.

EXAMPLE 3 EC 495 was grown in Nutrient Dextrose Broth (NDB) andmaintained under rotary agitation on a rotary shaker at 220 RPM at 30 C.for 24 hours. The culture was then centrifuged for 15 minutes at 9,000RPM to separate cells from supernatant. The supernatant was removed andsaved, being careful to avoid any visible retention of cells in thesupernatant. The cellular pellet was then resuspended in 0.85 percentphysiological saline and centrifuged as above while discarding thesupernatant. This was twice repeated to eliminate culture supernatantfrom the cell fraction. The cells were then resuspended in physiologicalsaline.

Seventy-five grams of burley stem were divided equally into three 25gram parts, as in Example 2. Parts A, B, and C were added separately to250 ml aliquots of water in 500 ml Erlenmeyer flasks, wherein themixture pH was 5.2 to 5.4. The supernatant from the first centrifugalseparation was added in a 25 ml quantity to Part B. The resuspendedcellular pellet was readjusted to its pre-centrifuged volume and 25 mlwas added to Part A. Neither cells nor supernatant were added to Part C.Parts A, B, and C were then held under agitation at 220 RPM (equipmentas in Example 2) for 24 hours at 30 C. After 24 hours incubation, thefollowing was observed:

Part

General Appearance Quality of Stem 0 Hours 24 Hours Digestion A wholestem viscous Excellent No intact whole stern whole stem slightly Poorviscous Many intact whole stems C whole stem whole stern None All wholestem At termination the pH of parts A and B were 6.87.2 while that ofPart C was 5.8.

EXAMPLE 4 25 gram portions each of flue-cured fines, burley fines,turkish tobacco, manufacturing fines and winnowers were separately addedto 250 ml quantities of tap water as in Example 2. A culture of EC 495grown as in Example 3 was separated into 6-25 ml quantities. Eachtobacco-water mixture was then inoculated with 25 ml each of EC 495culture. All flasks were agitated as in Example 3. All mixturesexhibited a viscous appearance in which excellent digestion hadoccurred. Subsequently, each mixture was hand cast as follows: A mlquantity of each treated mixture was mixed in a quart size Waringblender (Model 5011) cup with .100 ml of water for 3 minutes to obtain aslurry. Separately each slurry was then evenly spread to dry on astainless steel sheet mounted over a steam bath. After drying, none ofthe mixtures as cast sheets exhibited large fiber bundles.

EXAMPLE 5 240 grams each of flue-curedand burley stems, each from acommon batch, were added to four separate containers (A, B, C and D) inwhich 6 liters of tap water had been placed. 600 ml of EC 495 inoculumprepared as in Example 3 was added to each container. The followingconditions applied to the containers:

Treat- Con- Internal Shaft Internal Air Ring ment tainer Agitation (RPM)Aeration (L/min) Temp.

A 680 None 30 B None 30 C 680 4.5 30 D 680 9.0 30

Initial pH of all mixtures was 5.4. After 6 hours the following wasobserved:

Quality of Stem Container pH General Appearance Digestion A 6.8Moderately viscous Good B 6.2 Slightly viscous Fair-Good C 6.7Moderately viscous Good D 6.8 Viscous Excellent Hand cast sheets,prepared as in Example 4 indicated greatest whole stem degradation to bein Container D. The best quality cast sheet was also made from materialsout of Container D.

EXAMPLE 6 Hurley and flue-cured stem were treated as in Example 5 exceptthat the pH of the stem-water mixture in Containers A and B was elevatedfrom 5.3 to 7.0 with NH OH, prior to addition of EC 495 inoculum.Tobacco materials in C and D were not pH altered. The followingdigestion occurred by the fourth hour of treatment:

A stainless steel cylindrical tank was charged with 298 pounds water,26.56 pounds of burley stem and 33.2 pounds of EC 495 culture. The EC 4%culture was prepared as in Example 2 and was increased in volume bytransfer to 6 liter flasks of NDB. The 6 liter flasks contained 3,000 mlof NDB and, subsequent to inoculation, were incubated at 30 C. whilebeing maintained on a rotary shaker at 120 RPM prior to use on stem. Thetank contents, with an initial pH of 5.4 were agitated at 11 1 RPM andaerated at 9 liters/minute for 16 hours. Agitation was accomplished withan internal marine blade mounted'on a stainless shaft driven with aLlGHTNlN Mixer" (Model NDIA) and aeration was achieved with a multipleoutlet circular stainless tubing placed at the tank bottom. At the 14thhour digestion of stem material was complete as indicated by the highviscosity of the medium and the excellent quality of stem breakdown.

EXAMPLE 3 Tobacco stem digestion was accomplished as in Example 2,employing as inoculum other species which are pectolytic enzymeproducing microorganisms. inoculum was prepared as in Example 1. Thesestrains are Bacillus polymyxa ATCC 842 and Streptomyces cellulosae ATCC3313.

EXAMPLE 9 Stem digestion by other Erwini'a strains was accomplished asfor EC 495 in Example 1 except that 25 g each of flue-cured and burleystem were treated. These strains are Erwinia aroideae (ATCC Nos. 12286and 12312), Erwinia carotovora (ATCC Nos. 138, 17799, 8061 and 15713)and Erwinia atroseptica (ATCC No. 4446). E. aroideae (ATCC Nlo. 12286)and E. carotovora (ATCC No. 15713) exhibit digestion capabilitiesclosely approximating those of EC 495 with the former being best.

EXAMPLE 1'!) lnoculum levels from 5 to 20 percent concentrations byvolume were employed inthe digestion of burley: flue-cured sternmixtures. Conditions of these experiments were as for Example 4 exceptthat agitation of all mixtures was at 680 RPM and aeration of allmixtures was at 9 L/min. The following table illustrates the effect ofdiffering inoculum levels over 7.5 hours:

Time (hrs) lnoculum Quality of Stem Digestion 0 5 None 20 None 4 5 Fair20 Excellent 7.5 5 Excellent The higher the initial inoculum the lesstime required for digestion to take place.

EXAMPLE ll Concomitant with digestion of burley: flue-cured stemmixtures over a 7.5 hour treatment period with EC 495, the nitrate levelof stem slurry was decreased with increasing inoculum levels. For aninitial nitrate level of 4.63 percent the following changes wereobserved at 7.5 hours.

lnoculum Level Nitrate 5% 2.49 10% 1.26 15% 0.68 20% 0.45

EXAMPLE 12 Burley: flue-cured stem were treated as in Example S-D atvarying stem percent levels. The following table summarizes the stemlevels successfully treated over 7.5 hours:

Quality pf Stem viscous These data show that the higher percent levelsof tobacco solids in the tests above are not completely digested in 7.5hours but even at 13 percent solids, the digestion is of good quality.

EXAMPLE 13 Flue-cured and burley stems were treated as in Example 5 overa 16-hour period at an agitation rate of 380 RPM and without additionalaeration. The following table indicates reductions in the nitratefraction of the mixture.

Flue-cured and burley stems were treated as in Example 13 but in 1,200 gquantities each. The following table illustrates the nitrate reductionsachieved.

Time (hrs) Nitrate 4.58 16 0.51

As noted from the examples herein, aeration of the inoculated tobaccomixture together with agitation not only accelerates the digestion ordisintegration of the plant parts but it also makes them more suitablefor preparing reconstituted tobacco sheet of good quality forincorporation into tobacco products. It has also been found that themicrobial digestion significantly reduces the nitrate content of tobaccoand maintenance of a pH of about 7 is optimum for tobacco mixtures.

The following are examples of pilot plant runs in which the tobaccomixture after inoculation was continuously aerated and agitated untildigestion was considered completed. A total of 1,750 lbs. of materialwas treated consisting of 1,480 lbs. of water, 70 lbs. of fluecuredstems, 50 lbs. of burley stems and 150 lbs. of inoculum (12 lbs. ofwhich was burley stems). The mixture of materials was treated in a 290gallon tank fitted with an aeration line and an overhead mounted motorwith center drive agitation to facilitate uniform suspension of the stemmaterial and accelerate disintegration thereof. The agitation wasmaintained at 260 RPM and air bubbled through the mixture at a rate of 8cubic feet per minute. After the microbial action was completed theviscous fibrous mass was passed through a wet hammermill fitted withsizing screens of 0.016 and 0.012 inch openings. 3 percent by weight ofglycerine was added and the mixture was then made into reconstitutedtobacco. The enzyme activity, viable bacterial count and pH is set forthin the table below with the physical properties of the slurry and sheetafter a hour treatment.

MlCROBlOLOGICAL PROPERTIES OF THE SLURRY AND PHYSICAL PROPERTIES OF THESLURRY AND SHEET Microbiological Properties of the Slurry ChangeSampling Pectinase In Pectin Viable Time Activity Solution Bacterial(Hours) pH (secs) Viscosity Count l0) 0 Before 5.2 23.0 0 0.125 lnoculum6.4 4.5 80 7,800.0 0 After 5.8 8.1 64.8 780.0 1 Hr. after 5.8 7.6 66.0880.0 2 Hrs. after 6.0 8.9 61.0 930.0 3 Hrs. after 6.1 7.2 69.0 1,040.04 Hrs. after 6.3 7.3 68.0 1,240.0 5 Hrs. after 6.3 6.8 70.0 1,890.0

DETERMINATION OF PECTINASE ACTIVITY 1. A tobacco slurry is spun down ina centrifuge. The liquid is retained and the pH is adjusted to 8.0. A5.0 ml quantity is added to a 20 m1 pectin solution prepared indeionized water on an 8g/liter basis and ad justed to a pH of 8.0. Ameasured volume of the mixture is drawn into a calibrated pipette andits descent is timed (secs). The speed of descent is compared to that ofa standard pectin-water (20 ml;5 ml) mixture. Speed of descent is ameasure of solution viscosity reflecting the degradation of the pectinpolymer. A short (5.0-6.0 sec.) descent time represents high pectinaseactivity while a longer time (20-25 sec.) reflects lower activity. Thisprocedure is repeated after the pectinwater mixture stands for 0, 5 and10 minutes.

2. Viscosity of water is 5, therefore, a percent change greater thanapproximately percent is not possible.

Physical Properties of the Slurry & Sheet Brookfield Minus Mesh Consist-Stem Disin- Viscosity US. Standard ency tegration Centipoises SieveScreen (wt/wt.)

52,400 2.63 6.7 Good- Excellent Cast Sheet (5 Hour Treatment) ThicknessNet Tensile Density Flex (0.001") (gm/mm) (gm/cc) [f(200)] Reconstitutedsheets manufactured by the method described above hereinafter referredto as the microbial sheet, were compared with reconstituted tobaccosheet material made according to the process described above but withoutthe microbial treatment hereinafter referred to as the control sheet.Two identical samples of tobacco material were placed in a 43 gallontank which was fitted with an agitator as described above and aeratedwith 8 cubic feet of air per minute. The tobacco mixtures were treatedfor 16 hours after inoculation with EC 495. After 16 hours the treatedmixture was added to other tobacco materials and the mixture was passedthrough 0.016 to 0.012 inch screens of a wet hammermill. The mixture wasdivided into two equal parts, part A having 2 percent glycerin added,and part B having 4 percent glycerin added. The two parts were cast intosheets and evaluated separately. The control sheet was treated similarlyto the microbial sheet but without the use of inoculum.

The physical properties of the control and microbial treatedreconstituted tobacco were measured by normal techniques and are shownin the table below:

Comparative Physical Properties of Pilot Plant Sheets ProductionGlycerin Net l'lcx 'lhiekness Density Method Tensile f(200g) (0.001")(g/cc) tit/ Control 2 146 33 18.5 0.48

(without Bacterial tmt) 4 117 75 19.3 0.46

Bacterial 2 239 268 7.7 0.55

Treatment (Experimental) 4 310 607 11.8 0.68

The sheet material made according to the control procedure and the sheetmaterial .made according to the microbial treatment were incorporatedwith production shredded tobacco and sample cigarettes made therefrom.The cigarettes were smoked and evaluated by a smoke panel. It was foundthata noticable smoothing out of the tobacco smoke was observed in thecigarettes containing microbial treated reconstituted tobacco whencompared with the control cigarettes. Also, the pH of the smoke aerosolofthe cigarettes containing microbial treated reconstituted tobacco wasmore alkaline than the smoke of the control cigarettes.

Although this invention has been described with respect to the preferredembodiments, changes and modifications can be made which are within thefull scope of this invention.

We claim:

l. A method of forming a reconstituted tobacco sheet employing(microbial digestion of tobacco) material which comprises:

A. Subjecting a water slurry of tobacco material to the action of aquantity of pectolytic enzymeproducing microorganisms selected from theclass consisting of Erwinia carotovora, Erwinia atrseptica, Erwiniaaroideae, Bacillus polymyxa and Streptomyces cellulosae, saidmicroorganisms being dispersed in said slurry in concentrations of from5 to 20 percent by volume, and maintaining said microorganism and slurryat a pH between 5.2 and 8.5 and a temperature of from 24 to 400 C for upto 24 hours while aerating and agitating the slurry, to allow themicroorganisms to grow and act on said tobacco until the pectin isbroken down so as to form a thicker slurry of fibrous tobacco material,the fibers of the tobacco material being substantially unaffected by themicroorganism in the slurry;

B. Heating said slurry to a temperature sufficient to terminate theactivity of the microorganism; and

C. Casting the entire slurry mixture and then drying into areconstituted tobacco sheet.

2. The method of claim 1 wherein the plant slurry is maintained between2 and 14 percent solids content by weight.

3. The method of claim 1 wherein the pectolytic enzyme producingmicroorganism is Erwinia carotovora.

2. The method of claim 1 wherein the plant slurry is maintained between 2 and 14 percent solids content by weight.
 3. The method of claim 1 wherein the pectolytic enzyme producing microorganism is Erwinia carotovora. 